The emergence of antibodies, especially immobilized antibodies, as an article of commerce is an area whose continued expansion is virtually assured. Commercial uses rarely require the free antibody, but instead require the antibody bound to some insoluble substrate. This application relates to immobilized antibodies; more particularly, it relates to immobilized antibodies, especially immobilized monoclonal antibodies, intended for repeated commercial use.
Although the area of immobilized antibodies may seem analogous to the relatively well developed area of immobilized enzymes, the requirements and preparation of the two are not congruent. In particular, because an immobilized antibody may be used in a sterile and pyrogen-free environment, one of its requirements is that the underlying support matrix be capable of withstanding sterilization and depyrogenation procedures without affecting its ability to bind antibodies or the activity of the bound antibody. Since the antibody to be immobilized often is quite expensive it is also paramount that the immobilized antibody be capable of repeated use, leading to the second requirement that the antibody be covalently bound to the underlying support matrix. Another aspect of the efficient use of an antibody is the requirement that it exhibit activity and specificity when immobilized comparable to that manifested in solution, which leads to two derivative requirements. One is that the support to which the antibody is bound be chemically and biochemically inert as regards the antibody, the substrate, and other solution components in the environment of the process where used. The second derivative requirement is that the antibody be immobilized to the extent possible with both of its active (affinity) sites oriented away from the support, which provides for effective utilization of the antibody by allowing unhindered access of antigen to antibody. It is also important that the support components, binding agents, and antibody do not dissolve, wash off, or dissociate to any degree that might cause an immune response to these materials during subsequent use of the purified antigen. Lastly, the desire for a fixed bed process (or fluidized bed and similar processes) with a high linear velocity requires that the underlying matrix be noncompressible and attrition resistant.
We have discovered that an immobilized antibody system comprised of an aminated core support with an antibody covalently bonded thereto via amide linkages formed between the amino groups of the support and the carboxylic acid groups of the antibody, especially where the affinity sites of the antibody are oriented away from the support, meets all the aforementioned requirements. Therefore, such a system is highly advantageous in its field of intended use. Although the underlying support matrix is distantly related to that of U.S. Pat. No. 4,141,857, its application to a polypeptide or proteinaceous material by formation of amide linkages is hitherto neither disclosed nor appreciated, especially in the context of the aforementioned requirements.
Covalently bonded, immobilized antibodies are amply exemplified in the prior art. U.S. Pat. No. 4,399,217 relates to an immobilized antibody covalently bonded via diazo groups originating from diazotized poly(aminostyrene). U.S. Pat. No. 4,381,291 exemplifies covalent bonding of an antibody to a cyanogen bromide activated microcrystalline cellulose. In U.S. Pat. No. 4,357,311 there is described an antibody covalently bonded to trichloro-s-triazene activated microporous cellulose esters. In U.S. Pat. No. 4,347,312 the patentee immobilized antibodies by covalently bonding them to acylated aminopropylsilylated glass. U.S. Pat. No. 4,260,678 broadly discloses immobilization of antibodies, including the use of metal oxides as inorganic carriers. However, it is essential to recognize that all of the aforementioned art, which is exemplary only, relate solely to single use utilization, i.e., the immobilized antibody is used in a single immunochemical determination and then discarded. Furthermore, such art relate only to antibodies coupled in non-specific orientations, and do not teach sterilizability or nonpyrogenicity of the activated support. Hence, such art is of questionable relevance to the subject matter here where the repeated use of an immobilized antibody, preferably sterile and pyrogen-free, with little or no diminution of activity is an essential requirement.
In U.S. Pat. No. 4,361,509 the patentee coupled a monoclonal antibody in a non-specific orientation to a cyanogen bromide activated, crosslinked agarose to afford a column used in affinity chromatography. Arguably, this exemplifies a multiple or repeated use of a covalently bonded immobilized antibody but fails to address other features mentioned in the preceding paragraph.